Name: measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex
Uploaded: 2021-08-02T15:00:00
Description: Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

我们感谢威廉·桑德基金会（2017.008.02），动态系统中心（CDS），由欧盟计划ERDF（欧洲区域发展基金）和DFG（LA 2386）资助，以支持我们的工作。我们感谢Karina Guttek支持我们的实验。我们感谢Dirk Reinhold教授（OvGU，Magdeburg）为我们提供了原代T细胞。

T细胞实验根据伦理协议42502-2-1273 Uni MD进行。
1. 为实验准备细胞
注意：这种免疫沉淀的平均细胞数为1×107。 贴壁细胞必须在实验前一天接种，以便在实验当天有1×10个7 个细胞。
<ol><li>为实验准备贴壁细胞<ol>
<li>在实验开始前一天，将106 个贴壁细胞×10个6个贴壁细胞在20 mL培养基中（参见成分 材料表 ），用?...

<ol>
	<li>Lavrik, I. N., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CD95/Fas+signaling+at+the+DISC.">Regulation of CD95/Fas signaling at the DISC.</a> Cell Death and Differentiation. 19 (1), 36-41 (2012).</li><li>Krammer, P. H., Arnold, R., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+and+death+in+peripheral+T+cells.">Life and death in peripheral T cells.</a> Nature Reviews Immunology. 7 (7), 532-542 (2007).</li><li>Dickens, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+death+effector+domain+chain+DISC+model+reveals+a+crucial+role+for+caspase-8+chain+assembly+in+mediating+apoptotic+cell+death.">A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.</a> Molecular Cell. 47 (2), 291-305 (2012).</li><li>Fu, T. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+structure+of+caspase-8+tandem+DED+filament+reveals+assembly+and+regulation+mechanisms+of+the+death-inducing+signaling+complex.">Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.</a> Molecular Cell. 64 (2), 236-250 (2016).</li><li>Scaffidi, C., Medema, J. P., Krammer, P. H., Peter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLICE+is+predominantly+expressed+as+two+functionally+active+isoforms,+caspase-8/a+and+caspase-8/b.">FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b.</a> Journal of Biological Chemistry. 272 (43), 26953-26958 (1997).</li><li>Fox, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+structural+analysis+of+FADD:Caspase-8+complexes+defines+the+catalytic+dimer+architecture+for+co-ordinated+control+of+cell+fate.">Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.</a> Nature Communications. 12 (1), 1-17 (2021).</li><li>Schleich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+the+CD95+death-inducing+signaling+complex:+experimental+and+modeling+evidence+for+a+death+effector+domain+chain+model.">Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.</a> Molecular Cell. 47 (2), 306-319 (2012).</li><li>Hughes, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+death-inducing+signaling+complex+reveals+a+substrate+switch+that+determines+CD95-mediated+death+or+survival.">Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.</a> Molecular Cell. 35 (3), 265-279 (2009).</li><li>Lavrik, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+active+caspase-8+heterotetramer+is+formed+at+the+CD95+DISC+[2].">The active caspase-8 heterotetramer is formed at the CD95 DISC [2].</a> Cell Death and Differentiation. 10 (1), 144-145 (2003).</li><li>Hoffmann, J. C., Pappa, A., Krammer, P. H., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+C-terminal+cleavage+product+of+procaspase-8,+p30,+defines+an+alternative+pathway+of+procaspase-8+activation.">A new C-terminal cleavage product of procaspase-8, p30, defines an alternative pathway of procaspase-8 activation.</a> Molecular and Cellular Biology. 29 (16), 4431-4440 (2009).</li><li>Golks, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+CAP3+in+CD95+signaling:+New+insights+into+the+mechanism+of+procaspase-8+activation.">The role of CAP3 in CD95 signaling: New insights into the mechanism of procaspase-8 activation.</a> Cell Death and Differentiation. 13 (3), 489-498 (2006).</li><li>Ozt&#252;rk, S., Schleich, K., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+FLICE-like+inhibitory+proteins+(c-FLIPs):+Fine-tuners+of+life+and+death+decisions.">Cellular FLICE-like inhibitory proteins (c-FLIPs): Fine-tuners of life and death decisions.</a> Experimental Cell Research. 318 (11), 1324-1331 (2012).</li><li>Golks, A., Brenner, D., Fritsch, C., Krammer, P. H., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-FLIPR,+a+new+regulator+of+death+receptor-induced+apoptosis.">c-FLIPR, a new regulator of death receptor-induced apoptosis.</a> Journal of Biological Chemistry. 280 (15), 14507-14513 (2005).</li><li>Hughes, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-operative+and+hierarchical+binding+of+c-FLIP+and+caspase-8:+A+unified+model+defines+how+c-FLIP+isoforms+differentially+control+cell+fate.">Co-operative and hierarchical binding of c-FLIP and caspase-8: A unified model defines how c-FLIP isoforms differentially control cell fate.</a> Molecular Cell. 61 (6), 834-849 (2016).</li><li>Hillert, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+and+short+isoforms+of+c-FLIP+act+as+control+checkpoints+of+DED+filament+assembly.">Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.</a> Oncogene. 39 (8), 1756-1772 (2020).</li><li>Yu, J. W., Jeffrey, P. D., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+procaspase-8+activation+by+c-FLIPL.">Mechanism of procaspase-8 activation by c-FLIPL.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (20), 8169-8174 (2009).</li><li>Micheau, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long+form+of+FLIP+is+an+activator+of+caspase-8+at+the+Fas+death-inducing+signaling+complex.">The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.</a> Journal of Biological Chemistry. 277 (47), 45162-45171 (2002).</li><li>Chang, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-FLIPL+is+a+dual+function+regulator+for+caspase-8+activation+and+CD95-mediated+apoptosis.">C-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis.</a> EMBO Journal. 21 (14), 3704-3714 (2002).</li><li>Hillert, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+DISC+regulation+via+pharmacological+targeting+of+caspase-8/c-FLIPL+heterodimer.">Dissecting DISC regulation via pharmacological targeting of caspase-8/c-FLIPL heterodimer.</a> Cell Death and Differentiation. 27 (7), 2117-2130 (2020).</li><li>Fricker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+dissection+of+CD95+signaling+dynamics+reveals+both+a+pro-+and+antiapoptotic+role+of+c-FLIPL.">Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL.</a> Journal of Cell Biology. 190 (3), 377-389 (2010).</li><li>Arndt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+TCR+activation+kinetics+in+primary+human+T+cells+upon+focal+or+soluble+stimulation.">Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation.</a> Journal of Immunological Methods. 387 (1-2), 276-283 (2013).</li><li>Pietkiewicz, S., Eils, R., Krammer, P. H., Giese, N., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+treatment+of+CD95L+and+gemcitabine+in+pancreatic+cancer+cells+induces+apoptotic+and+RIP1-mediated+necroptotic+cell+death+network.">Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network.</a> Experimental Cell Research. 339 (1), 1-9 (2015).</li><li>Schleich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+the+DED+chains+at+the+DISC:+regulation+of+procaspase-8+activation+by+short+DED+proteins+c-FLIP+and+procaspase-8+prodomain.">Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.</a> Cell Death and Differentiation. 23 (4), 681-694 (2016).</li><li>Lavrik, I. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CD95+threshold+signaling:+Triggering+of+CD95+(FAS/APO-1)+at+low+concentrations+primarily+results+in+survival+signaling.">Analysis of CD95 threshold signaling: Triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling.</a> Journal of Biological Chemistry. 282 (18), 13664-13671 (2007).</li><li>Sprick, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-10+is+recruited+to+and+activated+at+the+native+TRAIL+and+CD95+death-inducing+signalling+complexes+in+a+FADD-dependent+manner+but+can+not+functionally+substitute+caspase-8.">Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8.</a> EMBO Journal. 21 (17), 4520-4530 (2002).</li><li>Neumann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+within+the+CD95+death-inducing+signaling+complex+decide+life+and+death+of+cells.">Dynamics within the CD95 death-inducing signaling complex decide life and death of cells.</a> Molecular Systems Biology. 6 (1), 352 (2010).</li><li>Kischkel, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity-dependent+APO-1+(Fas/CD95)-associated+proteins+form+a+death-inducing+signaling+complex+(DISC)+with+the+receptor.">Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.</a> EMBO Journal. 14 (22), 5579-5588 (1995).</li><li>Seyrek, K., Richter, M., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+sweet+regulation+of+apoptosis:+the+role+of+glycosylation+and+galectins+in+apoptotic+signaling+pathways.">Decoding the sweet regulation of apoptosis: the role of glycosylation and galectins in apoptotic signaling pathways.</a> Cell Death and Differentiation. 26 (6), 981-993 (2019).</li><li>Kallenberger, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-+and+interdimeric+caspase-8+self-cleavage+controls+strength+and+timing+of+CD95-induced+apoptosis.">Intra- and interdimeric caspase-8 self-cleavage controls strength and timing of CD95-induced apoptosis.</a> Science Signaling. 7 (316), 23 (2014).</li></ol>

这种方法首先由Kischkel等人27 描述，并从那时起由几个小组成功开发。为了在该复合物中进行有效的DISC免疫沉淀和监测半胱天冬酶-8处理，必须考虑几个重要问题。
首先，在免疫沉淀期间必须遵循所有洗涤步骤。特别重要的是琼脂糖珠的最终洗涤步骤和蜂蜡珠的干燥。必须正确完成此操作以增加免疫沉淀的信噪比，从而允许在DISC上检测caspase-8募集和处理。?...

研究最好的死亡受体（DRs）之一是CD95（Fas，APO-1）。外源性凋亡途径始于DR与其同源配体的相互作用，即CD95L与CD95相互作用或TRAIL与TRAIL-Rs结合。这导致在相应的DR处形成DISC，DISC由CD95，FADD，procaspase-8 / -10和c-FLIP蛋白1，2组成。此外，DISC是通过含死亡域（DD）的蛋白质（例如CD95和FADD）与含DED的蛋白质（例如FAD，procaspase-8 / -10和c-FLIP）之间的相互作用组装的（图1）。Procaspase-8通过其DED的结合经历寡聚化，导致DED细丝的形成，然后Procaspase-8的激活和处理。这触发了半胱天冬酶级联反应，导致细胞死亡（图1）3、4。因此，procaspase-8是由CD95或TRAIL-Rs介导的外源性凋亡途径的中枢引发子半胱天冬酶，在相应的大分子平台DISC上激活。
已知Procaspase-8的两种亚型，即procaspase-8a（p55）和-8b（p53），被招募到DISC5中。两种亚型均由两个DED组成。DED1和DED2位于procaspase-8a/b的N端部分，后跟催化p18和p10结构域。对procaspase-8 DEDs的详细冷冻电子显微镜（cryo-EM）分析揭示了Procaspase-8蛋白组装成称为DED细丝4，6的丝状结构。值得注意的是，线性procaspase-8链最初被认为参与二聚化，然后在DISC处进行procaspase-8激活。现在，已知这些链只是procaspase-8 DED细丝的子结构，后者由三个链组成三个螺旋3，4，6，7。
在DED灯丝处二聚化时，procaspase-8a / b的构象变化导致Procaspase-8活性中心的形成及其激活3，8。接下来是procaspase-8处理，其通过两种途径介导：第一种途径通过p43 / p41裂解产物的产生，第二种途径通过p30裂解产物的初始产生。p43 / p41途径由Asp374处procaspase-8a / b的裂解启动，导致p43 / p41和p12裂解产物（图2）。此外，这些片段在Asp384和Asp210/216处被自催化裂解，从而形成活性半胱天冬酶-8异四聚体p102/p1829，10，11。此外，研究表明，在p43 / p41处理途径的同时，procaspase-8a / b也在Asp216处被切割，这导致形成C端裂解产物p30，然后其蛋白水解至p10和p1810（图2）。
DED灯丝处的Procaspase-8a / b激活受到名为c-FLIPs12的蛋白质的严格调节。c-FLIP蛋白以三种亚型出现：c-FLIP Long（c-FLIPL），c-FLIP Short（c-FLIPS）和c-FLIP Raji（c-FLIP R）。所有三种亚型在其N端区域都包含两个DED。c-FLIPL还具有催化非活性的半胱天冬酶样结构域12、13。c-FLIP-c-FLIPS和c-FLIPR的短同种型都以抗凋亡的方式通过破坏DISC 6，14，15处的DED长丝形成而起作用。此外，c-FLIPL可以以浓度依赖性方式调节半胱天冬酶-8的活化。这可以导致促凋亡和抗凋亡作用16，17，18。通过形成催化活性的procaspase-8 / c-FLIPL异源二聚体，c-FLIPL导致procaspase-8活性中心的稳定及其活化。c-FLIPL的促凋亡或抗凋亡功能直接取决于其在DED细丝上的量以及随后组装的procaspase-8 / c-FLIPL异源二聚体19的量。DISC处的c-FLIPL浓度低或中等浓度导致DED长丝处产生足够量的procaspase-8 / c-FLIPL异源二聚体，这支持了Caspase-8的活化。相反，增加的c-FLIPL量直接导致其在DISC20处具有抗凋亡作用 。
综上所述，在DISC上procaspase-8a / b的激活和处理是一个高度监管的过程，涉及几个步骤。本文讨论了直接在DISC上测量procaspase-8处理以及分析该复合物的组成。这将使用CD95 DISC作为典型的DR复合物来呈现。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12.5% SDS gel</td>
			<td>self made</td>
			<td></td>
			<td>for two separating gels: 
			3.28 mL distilled H2O 
			2.5 mL Tris; pH 8.8; 1.5 M 
			4.06 mL acrylamide 
			100 &#181;L 10% SDS 
			100 &#181;L 10% APS 
			7.5 &#181;L TEMED 
			 
			for two collecting gels: 
			3.1 mL distilled H2O 
			1.25 mL Tris; pH 6.8; 1.5 M 
			0.5 mL acrylamide 
			50 &#181;L 10% SDS 
			25 &#181;L 10% APS 
			7.5 &#181;L TEMED</td>
		</tr>
		<tr>
			<td>14.5 cm cell dishes</td>
			<td>Greiner</td>
			<td>639160</td>
			<td></td>
		</tr>
		<tr>
			<td>acrylamide</td>
			<td>Carl Roth</td>
			<td>A124.1</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-actin Ab</td>
			<td>Sigma Aldrich</td>
			<td>A2103</td>
			<td>dilution: 1:4000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-APO-1 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by Enzo</td>
			<td>ALX-805-038-C100</td>
			<td>used only for immunoprecipitation</td>
		</tr>
		<tr>
			<td>anti-caspase-10 Ab</td>
			<td>Biozol</td>
			<td>MBL-M059-3</td>
			<td>dilution: 1:1000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-caspase-3 Ab</td>
			<td>cell signaling</td>
			<td>9662 S</td>
			<td>dilution: 1:2000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-caspase-8 Ab C15</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-804-242-C100</td>
			<td>dilution: 1:20 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-CD95 Ab</td>
			<td>Santa Cruz</td>
			<td>sc-715</td>
			<td>dilution: 1:2000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-c-FLIP NF6 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-804-961-0100</td>
			<td>dilution: 1:10 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-FADD 1C4 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ADI-AAM-212-E</td>
			<td>dilution: 1:10 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-PARP Ab</td>
			<td>cell signaling</td>
			<td>9542</td>
			<td>dilution: 1:1000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Carl Roth</td>
			<td>9592.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Carl Roth</td>
			<td>4227.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford solution 
			Protein Assay Dye Reagent Concentrate 450ml</td>
			<td>Bio Rad</td>
			<td>500-0006</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>CD95L</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-522-020-C005</td>
			<td></td>
		</tr>
		<tr>
			<td>chemoluminescence detector 
			Chem Doc XRS+</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protese Inhibitor Cocktail (PIC)</td>
			<td>Sigma Aldrich</td>
			<td>11 836 145 001</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>DPBS (10x) w/o Ca, Mg</td>
			<td>PAN Biotech</td>
			<td>P04-53500</td>
			<td>dilution 1:10 with H2O, storage in the fridge</td>
		</tr>
		<tr>
			<td>eletrophoresis buffer</td>
			<td>self made</td>
			<td></td>
			<td>10x electrophoresis buffer: 
			60.6 g Tris 
			288 g glycine 
			20 g SDS 
			ad 2 L H2O 
			1:10 dilution before usage</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1 HRP</td>
			<td>SouthernBiotech</td>
			<td>1070-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG2b</td>
			<td>SouthernBiotech</td>
			<td>1090-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG-HRP</td>
			<td>SouthernBiotech</td>
			<td>4030-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Interleukin-2 Human(hIL-2)</td>
			<td>Merckgroup/ Roche</td>
			<td>11011456001</td>
			<td>for activation of T cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Carl Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>loading buffer 
			4x Laemmli Sample Buffer,10 mL</td>
			<td>Bio Rad</td>
			<td>161-0747</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Luminata Forte Western HRP substrate</td>
			<td>Millipore</td>
			<td>WBLUFO500</td>
			<td></td>
		</tr>
		<tr>
			<td>lysis buffer</td>
			<td>self made</td>
			<td></td>
			<td>13.3 mL Tris-HCl; pH 7.4; 1.5 M 
			27.5 mL NaCl; 5 M 
			10 mL EDTA; 2 mM 
			100 mL Triton X-100 
			add 960 mL H2O</td>
		</tr>
		<tr>
			<td>medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine,&#160; 2,438 g/L</td>
			<td>PAN Biotech</td>
			<td>P04-41154</td>
			<td>adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium</td>
		</tr>
		<tr>
			<td>medium for primary T cells</td>
			<td>gibco by Life Technologie</td>
			<td>21875034</td>
			<td>adding 10% FCS and 1% Penicillin-Streptomycin to the medium</td>
		</tr>
		<tr>
			<td>milk powder</td>
			<td>Carl Roth</td>
			<td>T145.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Carl Roth</td>
			<td>P030.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td>self made</td>
			<td></td>
			<td>20x PBST: 
			230 g NaCl 
			8 g KCl 
			56.8 g Na2HPO4 
			8 g KH2PO4 
			20 mL Tween-20 
			ad 2 L H2O 
			dilution 1:20 before usage</td>
		</tr>
		<tr>
			<td>PBST + 5% milk</td>
			<td>self made</td>
			<td></td>
			<td>50 g milk powder + 1 L PBST</td>
		</tr>
		<tr>
			<td>PHA</td>
			<td>Thermo Fisher Scientific</td>
			<td>R30852801</td>
			<td>for actavation of T ells</td>
		</tr>
		<tr>
			<td>Power Pac HC</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Standard All Blue</td>
			<td>Bio Rad</td>
			<td>161-0373</td>
			<td>use between 3-5 &#181;L</td>
		</tr>
		<tr>
			<td>Protein A Sepharose CL-4B beads</td>
			<td>Novodirect/ Th.Geyer</td>
			<td>GE 17-0780-01</td>
			<td>affinity resin beads prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>scraper</td>
			<td>VWR</td>
			<td>734-2602</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth</td>
			<td>4360.2</td>
			<td></td>
		</tr>
		<tr>
			<td>shaker</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Carl Roth</td>
			<td>K305.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans Blot Turbo mini-size transfer stacks</td>
			<td>Bio Rad</td>
			<td>170-4270</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TransBlot Turbo 5x Transfer Buffer</td>
			<td>Bio Rad</td>
			<td>10026938</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TransBlot Turbo Mini-size nictrocellulose membrane</td>
			<td>Bio Rad</td>
			<td>170-4270</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Trans-Blot-Turbo</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Chem Solute</td>
			<td>8,08,51,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Pan Reac Appli Chem</td>
			<td>A4974,1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex

外源性细胞凋亡由死亡受体（DR）的激活介导，例如CD95 / Fas / APO-1或肿瘤坏死因子相关的凋亡诱导配体（TRAIL）受体1 /受体2（TRAIL-R1 / R2）。这些受体及其同源配体的刺激导致死亡诱导信号复合物（DISC）的组装。DISC包括DR，具有死亡结构域的适应蛋白Fas相关蛋白（FADD），procaspases-8/-10和细胞FAD样白细胞介素（IL）-1β-转化酶抑制蛋白（c-FLIP）。DISC用作procaspase-8处理和激活的平台。后者 通过其 在DISC上组装的死亡效应域（DED）细丝中的二聚/寡聚化发生。 
procaspase-8的激活之后是其处理，其分几个步骤进行。在这项工作中，描述了一个已建立的实验工作流程，该工作流程允许测量DISC形成和处理该复合物中的procaspase-8。该工作流程基于免疫沉淀技术，并辅以蛋白质印迹分析。该工作流程允许仔细监测PROCASPASE-8募集到DISC及其处理的不同步骤，并且与研究外源性凋亡的分子机制高度相关。

为了分析CASPASE-8募集到DISC及其在CD95 DISC上的处理，本文描述了一种经典的工作流程，该工作流程将CD95 DISC的IP与蛋白质印迹分析相结合。这允许在DISC上检测半胱天冬酶-8激活的几个关键特征：半胱天冬酶-8激活大分子平台的组装，Procaspase-8向DISC的募集，以及该引发子半胱天冬酶的处理（图1和图2）。该工作流程涉及以时间依赖性方式用CD95L处理敏感细胞...

Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

在这里，提出了一种实验工作流程，可以直接在死亡诱导信号复合物（DISC）上检测caspase-8处理并确定该复合物的组成。这种方法具有广泛的应用，从解开细胞死亡途径的分子机制到细胞凋亡网络的动态建模。

测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

凋亡的开始是通过在标记分子平台上激活半胱天冬酶介导的。我们提出了关键起始细胞死亡复合物DISC的检测，其作为半胱天冬酶-8激活的平台。该技术的优点是它允许检测DISC复合物的组装和组成，同时提供有关该复合物中caspase-8处理的信息。用80%至90%汇合并粘附在培养皿上的细胞开始实验。丢弃培养基并将新鲜培养基加入贴壁细胞中。然后通过以一定角度保持板并将配体移液到培养基中而不接触贴壁细胞来刺激具有选定浓度的CD95L的细胞。要收获细胞，请将细胞培养皿放在冰上。向细胞悬浮液中加入10毫升冷PBS，并将附着的细胞从板上刮下。将细胞悬浮液收集在50毫升管中。用10毫升冷PBS洗涤细胞皿两次，并将洗涤溶液加入相同的50毫升管中。然后将细胞悬浮液以500倍G在四摄氏度下离心五分钟。弃去上清液后，将细胞沉淀重悬于一毫升冷PBS中。然后将细胞悬浮液转移到1.5毫升的管中。离心细胞悬浮液，并再次将细胞沉淀重悬于一毫升冷PBS中。再次重复离心，然后将细胞沉淀重悬于一毫升裂解缓冲液中，并在冰上孵育30分钟。孵育后，以最大速度在4摄氏度下离心裂解物15分钟，然后将上清液转移到干净的管中。丢弃沉淀并在另一个管中收集50微升裂解物样品以确定蛋白质浓度。对于免疫沉淀，向裂解物中加入2微升抗APO1抗体和10微升制备的蛋白A琼脂糖珠。将10微升的微升磁珠单独加入含有裂解物的单独管中，以用作微珠对照。将含有裂解物，抗体和蛋白质A的混合物在4摄氏度下孵育过夜，并轻柔混合。第二天，将混合物以500倍G在四摄氏度下离心四分钟。弃去上清液后，通过加入一毫升冷PBS洗涤珠子，并在离心后丢弃上清液。至少重复此步骤三次。丢弃最后一次PBS洗涤后，最好用50微升Hamilton注射器吸出珠子。要进行蛋白质印迹，向磁珠中加入20微升4X加载缓冲液，并在95摄氏度下加热10分钟。同时，将裂解物对照在95摄氏度下加热五分钟。将裂解物、免疫沉淀剂和蛋白质标准品上样到 12.5% SDS 凝胶上，并以 80 伏的恒定电压电泳。凝胶电泳完成后，将蛋白质从SDS凝胶转移到硝酸纤维素膜上。转移完成后，将转印膜放入盒子中，在轻柔搅拌下将其在封闭溶液中孵育一小时，然后用PBST洗涤膜三次，持续五分钟。为了检测蛋白质，将第一个一抗以指示的稀释液加入膜中，并在四摄氏度下以轻柔搅拌孵育过夜。第二天，用三次五分钟的PBST洗涤膜，然后用20毫升二抗孵育膜，在室温下轻轻摇动一小时。再次用PBST洗涤膜三次。丢弃最后一次PBST洗涤后，向膜中加入约一毫升辣根过氧化物酶底物并检测化学发光信号。用CD95L刺激宫颈癌HeLa-CD95细胞导致通过CD95免疫沉淀监测高水平的CD95 DISC形成。在这些CD95免疫沉淀中观察到CD95，FADD，procaspase-8，procaspase-10和c-FLIPS，表明DISC形成有效。在免疫沉淀中检测到p43-FLIP和p22-FLIP的裂解产物，表明半胱天冬酶-8活化。用于分析CD95 DISC形成的过度表达c-FLIP长的HeLa-CD95细胞。与亲本HeLa-CD95细胞类似，在没有CD95L处理的情况下，在这些细胞的免疫沉淀中未检测到FAD，procaspase-9，procaspase-10，c-FLIP蛋白和PARP1的募集。活化的原代T细胞在抗CD95免疫沉淀物中具有高水平的CD95，FADD，procaspase-8，procaspase-10和c-FLIPS。该技术允许分析分子DISC的形成，包括在DISC处理CASPASE-8。所有洗涤步骤都非常重要。此外，应认真对待刺激点和潜伏时间。这是测量DISC复合物上组件的唯一方法。然而，为了测量半胱天冬酶-8活化，还可以实施半胱天冬酶-8活性测定。这种方法使我们能够获得对细胞凋亡起始的新见解。

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

酶的结晶和结构确定：多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...